Antibacterial surface of metal-organic framework-chitosan composite films

ABSTRACT

A substrate having an antibacterial surface includes a chitosan matrix and water-stable metal-organic frameworks dispersed throughout the chitosan matrix. The water-stable metal-organic frame-works are present in an amount of 5% wt/wt to 20% wt/wt based on total solids of the substrate

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/531,183, filed Jul. 11, 2017, which is herein incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under a NSF CAREER Award(award number 1352201) awarded by the National Science Foundation. Thegovernment may have certain rights in the invention.

BACKGROUND

The prevalence of antibiotic resistant bacteria poses a serious threatto human health, leading to increased and prolonged bacterialinfections. While bacteria in the free-floating, planktonic state remainsusceptible to traditional antibiotics, the vast majority of bacteriaexist in the biofilm state, where many antimicrobial agents are lesseffective. The Gram-negative bacterium Pseudomonas aeruginosa (P.aeruginosa) is one particularly concerning bacterial strain due to itscapacity to rapidly and efficiently form biofilms as well as itsinherent ability to develop resistance to antibiotics. The biofilm lifecycle is considered to occur in five stages, with the first two stepsconsisting of reversible and irreversible attachment of planktonicbacteria onto a surface. Therefore, identifying a material with theinherent properties to ultimately repel or reduce the bacterial adhesionof harmful pathogens represents a promising direction for controllingbiofilm formation.

SUMMARY

Example 1 is a substrate having an antibacterial surface. The substrateincludes a chitosan matrix and water-stable metal-organic frameworksdispersed throughout the chitosan matrix. The water-stable metal-organicframeworks are present in an amount of 5% wt/wt to 20% wt/wt based ontotal solids of the substrate.

In Example 2, the substrate of Examplel, wherein the water-stablemetal-organic frameworks are copper-based, water-stable metal organicframeworks.

In Example 3, the substrate of Example 1, wherein the water-stablemetal-organic frameworks are H₃[(Cu₄Cl)₃—(BTTri)₈](H₃BTTri=1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene).

In Example 4, The substrate of Example 1, wherein the water-stablemetal-organic frameworks are crystalline after 72 hours in a nutrientbroth media.

In Example 5, the substrate of Examplel, wherein the substrate is abiomedical substrate.

In Example 6, the substrate of Example 1, wherein the water-stablemetal-organic frameworks present in an amount of 5% wt/wt based on totalsolids of the substrate.

In Example 7, a method of making a substrate having an antibacterialsurface includes dispersing water-stable metal-organic frameworks in achitosan matrix to form a water-soluble chitosan/water-stablemetal-organic framework material, the water-stable metal-organicframeworks present in the water-soluble chitosan/water-stablemetal-organic framework material in an amount of 5% wt/wt to 20% wt/wtbased on total solids of the material, and converting the water-solublechitosan/water-stable metal-organic framework material to awater-insoluble chitosan/water-stable copper-based metal-organicframework material with a buffer solution.

In Example 8, the method of Example 7, wherein the water-stablemetal-organic frameworks are copper-based, water-stable metal organicframeworks.

In Example 9, the method of Example 7, wherein the water-stablemetal-organic frameworks are H₃[(Cu₄Cl)₃—(BTTri)₈](H₃BTTri=1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene).

In Example 10, the method of Example 7, wherein the water-stablemetal-organic frameworks are crystalline after 72 hours in a nutrientbroth media.

In Example 11, the method of Example 7, and further comprising formingthe water-insoluble chitosan/water-stable copper-based metal-organicframework material into a biomedical device.

In Example 12, the method of Example 7, wherein the water-stablemetal-organic frameworks present in an amount of 5% wt/wt based on totalsolids of the substrate.

In Example 13, a method of using a material to reduce adhesion ofbacteria on a surface of the material includes exposing the materialcomprising copper-based metal-organic frameworks dispersed throughout achitosan matrix to a solution containing the bacteria, wherein duringthe exposure the material reduces bacterial adhesion by at least 85% inthe first six hours of exposure as compared to material that does notinclude the copper-based metal-organic frameworks and wherein after thefirst six hours of exposure the material does not release copper in abactericidal effective amount.

In Example 14, the method of Example 13, and further including removingthe material from exposure to the bacteria, sterilizing the materialafter the removing step, and exposing the material to a new environmentof bacterial after the sterilizing step, wherein during the secondexposing step the material reduces bacterial adhesion by at least 85% inthe first six hours of exposure as compared to material that does notinclude the copper-based metal-organic frameworks and wherein thematerial is not subject to regeneration before the second exposing step.

In Example 15, the method of Example 13 wherein the bacteria isPseudomonas aeruginosa.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description, which shows and describesillustrative embodiments of the invention. Accordingly, the drawings anddetailed description are to be regarded as illustrative in nature andnot restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a are powder x-ray diffraction (pXRD) diffraction patterns ofchitosan films, chitosan/Cu—BTTri films and Cu—BTTri powder.

FIG. 1b are attenuated total reflection infrared spectroscopy (ATR-IR)analysis of chitosan films, chitosan/Cu—BTTri films and Cu—BTTri powder.

FIG. 2a is a scanning electron microscope (SEM) image of a chitosan filmaccording to some embodiments.

FIG. 2b and FIG. 2c are SEM images of chitosan/Cu—BTTri films accordingto some embodiments.

FIG. 2d is an SEM image of the chitosan/Cu—BTTri film with an x-rayanalysis (EDX) overlay of copper distribution according to someembodiments.

FIG. 3a and FIG. 3b are bar charts reporting cellular viability after 6hours and 24 hours of exposure according to some embodiments.

FIG. 4 shows pXRD diffraction patterns of chitosan/Cu—BTTri film priorto and following a bacterial assay according to some embodiments.

FIG. 5 is a bar chart reporting cellular viability after 6 hours and 24hours of exposure according to some embodiments.

DETAILED DESCRIPTION

Disclosed herein is a substrate material containing chitosan and awater-stable metal-organic framework such as Cu—BTTri(H₃[(Cu₄Cl)₃—(BTTri)₈](H₃BTTri=1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene)). In certainembodiments, the material is an antibacterial substrate or film whichmay be used in biomedical applications. Methods of making the materialare also described.

Chitosan is a polysaccharide derived from the biopolymer chitin and hasbeen utilized in multiple biological studies due to its overallbiocompatibility and biodegradability. It is composed of β-(1,4)-linkedglucosamine and N-acetyl glucosamine units and has been shown to havelittle to no toxic byproducts. Although there has been emphasis on theantibacterial nature of chitosan in solution against planktonicbacteria, another common use of chitosan as a biomaterial is in the formof wound dressings where it functions as a hemostatic agent. Thrombusformation arising from this type of hemostatic effect may increase thelikelihood of biofilm formation, as the adhered proteins onto thechitosan wound dressing provide an ideal area for which bacteria toattach. Therefore, embedding the chitosan matrix with a compound thatmay improve the materials ability to resist bacterial attachment is oneapproach to this challenge.

Metal-organic frameworks (MOFs) are a unique class of hybrid materialscombining metal centers with organic linkers to produce materials withhigh porosity. Variation of the metal and ligand has large effects onthe overall properties and, therefore, applications of MOFs. While thesematerials have been widely exploited in gas storage and catalysis, thereare fewer studies utilizing MOFs in biological settings. The knownbiocidal activity of copper has led to some investigation ofcopper-based MOFs in biological settings for use as potentialantibacterial agents. For example, a small number of initial bacteriastudies have been carried out using the copper-based MOF copper(II)benzene-1,3,5-tricarboxylate (also known as Cu—BTC or HKUST-1) as anantibacterial agent, attributing the observed biocidal effects to metalsites and the slow, continuous leaching of copper ions, as this MOFundergoes substantial and immediate degradation in aqueous systems.These particular studies did not evaluate bacterial attachment ontoMOF-containing surfaces, but rather the biocidal activity of the MOFagainst planktonic bacteria in solution by copper ions released from theframework. Indeed, copper leaching into bacterial solution hassignificant antibacterial effects, however in certain embodiments, it isdesirable to develop a material that intrinsically prevents theattachment of bacteria without the need for a biocide-eluting surface.

The material disclosed herein includes water-stable MOFs. For example,in some embodiments the MOF remains crystalline and intact after 72hours in a nutrient broth media as part of a 24-hour bacterialattachment experiment as described herein. In some embodiments, thewater-stable MOFs are copper-based. Cu—BTTri (H₃[(Cu₄Cl)₃—(BTTri)₈](H₃BTTri=1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene)) is an exemplarywater-stable MOF. CuBTTri is formed of [Cu₄Cl]⁷⁺ square planar unitsbound to BTTri³⁻ ligands. Each triazolate ligand interacts with sixcopper sites on [Cu₄Cl]⁷⁺ units. CuBTTri has increased metal-ligand bondstrength compared to other copper carboxylate MOFs, which confersgreater water stability.

The water-stable MOF is incorporated in chitosan. The water-stable MOFcan be dispersed throughout the chitosan matrix. The material is denotedas chitosan/water-stable MOF throughout this text, and chitosan/Cu—BTTriwhen the water-stable MOF is CuBTTri.

In some embodiments, the water-stable MOF is present at 5% wt/wt to 10%wt/wt or 20% wt/wt (based on total solids of the material). In someembodiments, the water-stable MOF is present at 5% wt/wt (based on totalsolids of the material).

The antibacterial activity of water instable copper-based MOFs has beenstudied. In such systems, the antibacterial activity can be attributedto the presence of leached copper in in solution. In contrast, theantibacterial activity achieved using a water-stable, copper-based MOFpresents a more passive approach to a MOF-polymer antibacterial surface.and the antibacterial activity of the water-stable system cannot beattributed to copper in solution. The water stability of Cu—BTTri andthe presence of copper centers makes this MOFs a particularly attractivepotential candidate for biological applications.

In some embodiments, the surface of the device is an antibacterialsurface that reduces or inhibits bacterial attachment. In someembodiments, the antibacterial surface reduces the bacterial attachmentof Pseudomonas aeruginosa. Such a device has far-reaching implications,as planktonic bacteria are far more susceptible to antimicrobials and,therefore, less of a concern than biofilm formation.

A method of making a substrate having an antibacterial surface includesdispersing water-stable metal-organic frameworks (MOFs) in chitosan toform a water-soluble chitosan/water-stable metal-organic frameworkmaterial. The MOFs can be uniformly dispersed throughout the chitosanmatrix.

To reduce the likelihood of MOF structural changes arising from exposureto sodium hydroxide, a buffer solution, such as mild pH 8 sodiumphosphate buffer solution, can be used to convert the water-solublechitosan into water-insoluble chitosan.

EXAMPLES

Embodiments of the present invention are further defined in thefollowing non-limiting Examples. It should be understood that theseExamples, while indicating certain embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theembodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims. Unless otherwise noted, all parts, percentages, andratios reported in the following examples are on a weight basis.

Materials

Low molecular weight chitosan (96% deacetylated) and copper(I) iodide(99.5%) were purchased from Sigma-Aldrich (St. Louis, Mo., USA).Phosphate buffered saline (PBS) tablets and copper(II) chloridedihydrate were obtained from EMD Chemicals (Gibbstown, N.J., USA).1,3,5-tribromobenzene (98%), trimethylsilylacetylene (98%),trimethylsilyl azide (94%), diethylamine (99%), were purchased from AlfaAesar (Ward Hill, Mass., USA). Deionized water (18.2 MΩ·cm) was preparedusing a Millipore Direct-Q water purification system.Bis(triphenylphosphine)palladium(II) dichloride (98%) was obtained fromTCI America (Portland, Oreg., USA). Chelex-100 Resin was purchased fromBio-Rad (Hercules, Calif., USA). Pseudomonas aeruginosa (PAO1) wasprovided by Dr. Brad Borlee at Colorado State University. Oxoid™nutrient broth media (NBM, OXCM0001B), Oxoid™ nutrient agar (NA,OXCM0003B), and sodium chloride were purchased from Fisher Scientific(Fair Lawn, N.J., USA). CellTiter Blue was purchased from Promega(Madison, Wis., USA). Ethanol was purchased from Pharmco-AAPER(Brookfield, Conn., USA). 24-well and 96-well tissue culture nontreatedplates were obtained from Corning (Corning, N.Y., USA).

Characterization Techniques

Images were taken at magnification values of 250x and 500x using a JEOLJSM-6500F scanning electron microscope with an accelerating voltage of10.0 kV and a working distance of 10.1 mm (JEOL USA Inc., Mass., USA)equipped with a Thermo Electron energy dispersive X-ray spectrometer(EDX). All samples were dehydrated and coated with 15 nm of Au. All datawas processed using TEAM Software. Powder X-ray diffraction (pXRD)measurements were carried out using a Bruker D-8 Discover DaVinci X-raydiffractometer (Bruker, Billerica, Mass., USA) with CuKα radiation(λ=1.5406 Å), and the resulting data was plotted as intensity vs. 2 θ inOrigin Pro. ATR-IR spectra were recorded in the range of 600-4000 cm⁻ ona Nicolet 6700 spectrometer (Thermo Electron Corporation, Madison, Wis.,USA). Elemental analysis was performed using ICP-AES provided by theColorado State University Soil, Water and Plant Testing Laboratory.

Abbreviations

The following abbreviations are used:

Cu—BTTri=H₃[(Cu₄Cl)₃—(BTTri)₈]

H₃BTTri=1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene

Synthesis of H₃[(Cu₄Cl)₃(BTTri)₈(H₂O)₁₂].72H₂O (Cu—BTTri-H₂O)

The triazole ligand 1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene (H₃BTTri)was synthesized following a previously reported protocol by A.Demessence, D. M. D'Alessandro, M. L. Foo, J. R. Long, J. Am. Chem.Soc., 2009, 131, 8784, which is herein incorporated by reference in itsentirety. In brief, H₃BTTri (225 mg) was suspended in DMF (10 mL) anddissolved at pH 4 following addition of 0.1 M hydrochloric acid.CuCl₂.H₂O (383 mg) was subsequently added directly to the solution anddissolved, and the resulting mixture was heated in a sealed vessel at100° C. for 3 days. The reaction produced a violet precipitate(Cu—BTTri-DNIF) that was isolated from the supernatant by centrifugationand washed thoroughly with DMF and Millipore water. The MOF wassuspended in deionized water and heated at 80° C. for 3 days to exchangeDMF, re-isolated by further centrifugation, then washed with Milliporewater to yield a light purple powder (Cu—BTTri-H₂O). IR: ν 3700-3000,3144, 2953, 1655, 1616, 1534, 1449, 1385, 1358, 1310, 1243, 1226, 1145,1100, 1024, 979, 885, 830, 775, 689, 678, 664 cm⁻¹.

Chitosan Acetate

Chitosan (2.5 g) was suspended in 1% acetic acid (100 mL) and stirreduntil dissolution. The resulting solution was frozen and lyophilized toobtain water-soluble chitosan acetate.

Chitosan Films

Chitosan acetate (180 mg) was dissolved with Millipore water (6 mL) (3%w v⁻¹). The solution was cast into a PTFE mold and the solvent wasallowed to evaporate over 48 hours. The resulting chitosan acetate filmwas removed and suspended in pH 8.0 250 mM sodium phosphate buffer (100mL). After 15 minutes, the buffer was exchanged and the suspensionrepeated, after which the film was washed with 5× Millipore water (100mL). 13 mm diameter films were prepared from the original material andused for subsequent experiments. IR: ν 3650-3000, 3355, 3278, 2917,2849, 1636, 1577, 1542, 1420, 1376, 1321, 1258, 1151, 1056, 1025, 894cm⁻¹.

Chitosan/Cu—BTTri Films

Chitosan/Cu—BTTri films were made having various Cu—BTTri content.Sample films having 20%, 10%, 5% and 1% Cu—BTTri wt/wt (relative tototal solids) were formed using the following process. The 10% Cu—BTTriwt/wt films were used in all experiments. The 20%, 5% and 1% samplefilms were synthesized for bacterial attachment studies. To reduce thelikelihood of MOF structural changes arising from exposure to sodiumhydroxide, a mild pH 8 sodium phosphate buffer solution was used toconvert the water-soluble chitosan acetate into insoluble chitosan.

Chitosan acetate was dissolved in Millipore water (6 mL) according toTable 1. Cu—BTTri was then added according to Table 1, and the viscousmixture was agitated to form a suspension. This suspension was cast intoa PTFE mold and allowed to evaporate over 48 hours. The resultingchitosan acetate/Cu—BTTri film was removed and placed in pH 8.0 250 mMsodium phosphate buffer (100 mL). After 15 minutes, the buffer wasexchanged and the process repeated, after which the film was washed with5× Millipore water (100 mL). 13 mm diameter films were punched from theoriginal material and used for subsequent experiments. IR: ν 3650-3000,3356, 3286, 2920, 2854, 1654, 1617 (Cu—BTTri), 1555, 1419, 1376, 1310,1247, 1227, 1148, 1064, 1023, 892, 826 (Cu—BTTri), 774 (Cu—BTTri) cm⁻¹.

TABLE 1 Chitosan acetate Cu-BTTri Sample (mg) (mg) 20% wt/wt 160 40 10%wt/wt 180 20  5% wt/wt 190 10  1% wt/wt 199 1

Chitosan and Chitosan/Cu—BTTri Cu Content and Soaking Studies

The average copper content of chitosan/Cu—BTTri films was determined byICP-AES analysis where dissolution of films (n=3) in 1% acetic acid wasperformed, followed by addition of 37% hydrochloric acid to decomposeCu—BTTri. The 10% wt/wt films were found to contain 295±8 μmol Cu/g.Additional formulations with 1, 5, and 20% Cu—BTTri wt/wt contained31±15, 156±12, and 527±97 μmol Cu/g, respectively. Chitosan filmsdissolved under identical conditions were found to contain 0.320±0.025μmol Cu/g. Based on the formula unit of Cu—BTTri-H₂O(H₃(Cu₄Cl)₃(BTTri)₈(H₂O)₁₂.72H₂O), the Cu—BTTri content of the finaldeprotonated 10% films was estimated at 11% wt/wt using the coppercontent determined by ICP-AES. In the case of 1, 5, and 20% wt/wt films,the estimated Cu—BTTri content was 1, 6, and 20% wt/wt, respectively.

In addition, films were analyzed for residual copper content fromsynthetic procedure by soaking in NBM at 37° C. for 24, 48, and 72hours. These solutions were analyzed for elemental analysis usingICP-AES. The resulting copper in solution from the chitosan andchitosan/Cu—BTTri films were determined after subtracting the coppercontent from the NBM itself under the same conditions. The averagecopper in solution (mg/L) was normalized for each film by the volume ofadded NBM (mL) and mass of each film (mg). The percent copper insolution was found by comparing the mass of copper from the soakingsolutions over the given soaking periods and the average mass of thetotal copper content of the films.

The soaking procedure revealed that a range of 0.76-2.47% copper wasfound in solution (relative to total amount of copper in films). Theresults from this analysis led to a post-synthetic pre-treatment stepwhere the films were immersed in NBM at 37° C. for 72 hours prior tobeginning bacterial attachment assays.

Characterization of Cu—BTTri and Chitosan/Cu—BTTri Films

Cu—BTTri was characterized by pXRD and found to be consistent with thepreviously reported diffraction pattern. Following the incorporation ofthe MOF into chitosan, the films were analyzed via pXRD (FIG. 1a ) andATR-IR (FIG. 1b ) to ensure that the Cu—BTTri remained structurallyintact. The pXRD spectrum of the chitosan/Cu—BTTri films demonstrate theretention of all major diffraction peaks originating from Cu—BTTri withoverlaps near 10 and 15-25 2 θ that were related to the chitosanmaterial. The ATR-IR spectrum show IR absorptions associated withCu—BTTri, present at 1617 (aromatic C═C stretch), 830, and 775 cm⁻¹ (C—Hout-of-plane bending), further supporting successful incorporation ofthe MOF.

The chitosan/Cu—BTTri materials were also examined by SEM-EDX to furtherevaluate the incorporation of Cu—BTTri into the chitosan films. FIG. 2ais a SEM image of a chitosan film; FIGS. 2b and 2c are SEM images of thechitosan/Cu—BTTri film; and FIG. 2d is an SEM image of thechitosan/Cu—BTTri film with an EDX overlay of copper distribution. Asshown in FIG. 2b , Cu—BTTri was directly embedded within the chitosanmatrix where it is observed that Cu—BTTri is present throughout theentire surface of the film. The material was then evaluated for theoverall distribution of copper by SEM-EDX using a copper analysis probe.FIG. 2d shows the copper overlay on the SEM image of thechitosan/Cu—BTTri film, where the overall distribution of copper isgenerally concentrated in areas that contain crystalline Cu—BTTri.

Bacterial Studies

Currently, there are a number of methods employed to decrease or haltthe adhesion step of bacteria onto a surface. The two main approachesare materials that release antibacterial agents and materials withbacteria killing or repelling surfaces. The first method is consideredan active approach, where the healthy bacteria are ultimatelycompromised by exposure to a biocidal agent being released from amaterial. Conversely, the second approach is considered passive, asthere is not a need for a drug-releasing agent, but rather the materialcontains inherent properties that reduce the amount of adhered bacteriaonto that surface (either through contact killing or repellingsurfaces). These passive surfaces are particularly attractive for use inbiomedical applications because they do not require a reservoir ofantibacterial agents and can theoretically be used multiple times. Thus,the chitosan/Cu—BTTri materials were tested to determine if they behaveas a passive antibacterial surface.

Additionally, the antibacterial nature of copper-based MOFs has beenprimarily investigated by exploiting the slow and steady release ofcopper ions into solution caused by the breakdown of water unstableMOFs. For example, it was previously observed that Cu—BTC grown on silkfibers exhibited high antibacterial action against both Escherichia coli(E. coli) and Staphylococcus aureus (S. aureus) planktonic bacteria. Ingeneral, copper ions have long been identified as an antibacterialagent, making it evident that copper-based MOFs are breaking down insolution over time and the copper ions are interacting with planktonicbacteria. In contrast, Cu—BTTri, has been shown to be stable in aqueousenvironments, such as phosphate buffered saline (PBS) and blood. Thus,the utilization of Cu—BTTri allows for the investigation of thepotential antibacterial nature of the MOF while eliminating orminimizing activity due to byproducts and possible leachates that couldbe causing the observed activity on planktonic bacteria.

Pseudomonas aeruginosa Bacteria Culture

An initial stock culture of P. aeruginosa (PAO1) was obtained bystreaking onto agar plates and inoculating a colony in NBM and grownovernight at 37° C. until an O.D._(600 mn)˜1.0 was reached. Thisbacterial solution was combined with glycerol (30% v/v) in a 1:1 fashionto obtain a final glycerol concentration of 15% (v/v). These solutionswere stored at −80° C. until use. Prior to each bacterial assay, afrozen culture was allowed to thaw and then centrifuged at 4700 rpm for10 minutes. The supernatant was discarded and the pellet was resuspendedusing 5 mL NBM. This was transferred to an additional NBM (45 mL) andallowed to grow overnight under stirring conditions until theO.D._(600 mn)˜1.0. The following day, the overnight culture was dilutedto an O.D._(600 mn)˜0.35 using warmed NBM prior to beginning theattachment assays.

Bacteria Attachment Assays

The ability of chitosan and chitosan/Cu—BTTri films (10% wt/wt) toinhibit bacterial attachment of P. aeruginosa over 6- and 24-hourexposure periods was assessed using two bacterial viability assays. Thisparticular bacteria strain is associated with a high level of antibioticresistance and is one of the most common strains associated with biofilmformation. Due to its ability to quickly form robust biofilms at woundsites, the ultimate goal is to find a material with the capabilities toinhibit the initial attachment of P. aeruginosa onto a surface,ultimately preventing the formation of a biofilm. This discovery couldhave significant impacts on the overall length and severity of bacterialinfections. Initial attachment experiments are performed after 6 hoursof exposure to a surface, as this is considered the most critical timeperiod after material implantation for biofilm formation to occur.However, it is imperative to ensure that inhibition is maintained overthe entire 24-hour challenge period.

Prior to bacteria attachment assays, all films were hydrated overnightusing sterile DI water before being transferred to vials containing amass normalized amount of NBM (1 mL NBM/3.05 mg film) as determined bythe leaching assays. The films were stored in NBM at 37° C. for 72hours. Before being exposed to the bacterial solution, the films wereremoved from the NBM and placed in a 24-well non-tissue culture treatedplate. An aliquot of P. aeruginosa bacterial solution (1 mL) was placedinto wells containing the films, with empty wells used as the positivecontrol. The wells were placed in an incubator at 37° C. for either 6 or24 hours before quantifying the attached bacteria.

A bacteria cellular viability assay was utilized to determine the amountof viable cells on the surface of the films or wells after the exposureperiod. This was done by removing the bacteria solution from all wells,washing the wells one time with sterilized PBS, moving the films to anew well such that only the bacteria attached to the films and not thesurrounding well was assayed, and CellTiter Blue solution (400 μL) wasadded. The CellTiter Blue solution consisted of 1:5 ratio of CellTiterBlue reagent with NBM. CellTiter Blue exploits the ability of healthybacteria to convert a blue colored compound resazurin (λ_(max)=600 nm)to the highly pink compound resorufin (X_(max)=570 nm). After additionof the solution, the plate was placed back in the 37° C. incubator for1-2 hours before 100 μL aliquots were transferred to a 96-well plate andthe absorbance was measured at 570 and 600 nm (Synergy 2 Multi-ModeReader, BioTek, Winooski, Vt., USA). The cellular viability obtained bythese absorbance values was normalized for film and well area. (n≥6)

By monitoring the absorbance features of both compounds, an overallincrease or decrease in metabolic activity of viable bacteria can beassessed by comparison to a positive control. In this case, the positivecontrol (PC) represents non-tissue culture treated polystyrene wells,however, throughout the text the chitosan films without theincorporation of Cu—BTTri will also be utilized and discussed as apositive control, as a further point of comparison. Regardless of theassignment of control wells, bacterial viability was assessed aftereither 6 or 24 hours and normalized by the given area available forbacteria attachment. FIG. 3a displays the results of this assay, withthe polystyrene well as the positive control (PC). After 6 hours ofexposure to bacteria, a 55-65% (displayed as the confidence interval)reduction in attachment is seen for the chitosan films, whilechitosan/Cu—BTTri films display an even greater reduction of 81-87% inattachment of viable bacteria. Given the established antibacterialproperties of chitosan, it is useful to consider the reduction in viablebacteria onto the chitosan/Cu—BTTri when compared to the chitosan itselfas a positive control. This results in a 50-68% reduction in attachmentafter the 6-hour period. This is a significant reduction to achieve in a6-hour exposure period, however it is important to ensure that theadherence of bacteria does not increase over 24 hours.

As shown in FIG. 3a , the ultimate reduction of attachment onto thechitosan/Cu—BTTri films is retained over the 24-hour period, with an82-86% reduction observed. Indeed, this is a substantial reduction toachieve given the bacteria strain of P. aeruginosa. In contrast, thereduction was not maintained for the chitosan films, with only 33-43%reduction of attachment remaining after 24 hours. If thechitosan/Cu—BTTri films are again compared to the chitosan filmsthemselves as the positive control, a 75-79% reduction in bacterialattachment onto chitosan/Cu—BTTri films is observed. All resultsdetermined by the CellTiter Blue assay were supported by enumerating thenumber of bacterial colonies on the films or wells using a sonicationand plating assay. This technique removes the viable bacteria from thesurface by sonicating for 30 minutes to liberate the attached bacteriaas the planktonic form, which can then be serial diluted and agar platedto ultimately determine the number of colony-forming units (CFUs).Again, the determined CFUs were normalized by the given attachment areaof the wells or films.

In addition, the number of colony-forming units (CFUs) that remained oneach 10% film or well after the exposure period was determined using asonication and plating method. The bacteria solution was removed fromall wells, the wells were washed one time with warmed NBM, films weretransferred to new wells, and fresh NBM was added to all wells. Theplate was then sonicated for 30 minutes to remove the viable bacteriafrom the films or wells and 100 μL aliquots were serial diluted and 50μL was plated on agar. The agar plates were placed in a static 37° C.incubator overnight and CFUs were counted the following morning. Theamount of attached bacteria identified using this method was normalizedby the area of the films or wells. (n=6)

Reusability of Chitosan and Chitosan/Cu—BTTri Films

The chitosan and 10% chitosan/Cu—BTTri films were saved after completingthe initial round of attachment studies to determine if a similarreduction in attachment could be seen with the sterilized films. Thiswas done by sterilizing the films in ethanol overnight and then allowingthem to rehydrate in water before performing the CellTiter Blue assayagain. This assay was performed in an identical fashion to initialbacterial attachment studies using CellTiter Blue and a 6- and 24-hourexposure period without the 72-hour NBM soaking period. (n=6)

FIG. 3b shows the results of this study, where it is seen that a similarreduction in attachment is seen for all samples and both time points forthe CellTiter Blue assay. Indeed, the bacteria cellular viabilitiesfound for chitosan and chitosan/Cu—BTTri in the second round of assaysis not statistically different from those determined from the firstround of assays at a 95% confidence level. This observed continuedfunction of the films demonstrates the usefulness and potentialreusability of these novel materials to be used as biomaterials forantibacterial applications. This also suggests that the films may indeedbe considered as passive antibacterial surfaces, as there is no loss offunctionality after initial exposure to bacteria.

Previous reports of P. aeruginosa bacterial adhesion onto a surface varywidely with regards to the percent reduction observed. An example of anapproach where an antimicrobial agent is released (in this case, nitricoxide) has been shown to inhibit P. aeruginosa attachment by 50-65%depending on the nitric oxide flux from the material. Another approachwhere contact killing is employed through the use of cationic peptidesimmobilized on a surface achieved up to 80% inhibition of P. aeruginosa.Finally, Hook et al. employed a passive approach for bacterial adhesionby implementing a novel material containing a combination of polymers toultimately achieve ˜80% reduction in P. aeruginosa attachment. Theseexamples highlight the significant reduction achieved in this work usingthe chitosan/Cu—BTTri materials, where an ˜85% reduction in achievedwithin the first 6 hours, maintained over 24 hours, and have thecapabilities to achieve the same reduction again after the initial roundof experiments.

Bacteria Control Studies (10% Chitosan/Cu—BTTri Films)

Due to potential antimicrobial effects from possible leachates from thechitosan/Cu—BTTri films (namely copper ions), a number of controlstudies were performed using P. aeruginosa both in solution and on thefilms themselves. Results from ICP-AES revealed that, for the 10% wt/wtfilms, 0.0725±0.0024 mg Cu/L NBM was present in the solutions after 72hours of soaking in NBM at 37° C., representing ˜2.5% of the totaltheoretical amount of copper in each film. To determine if this amounthad any bactericidal effects on P. aeruginosa, an equivalent amount wasintroduced to the bacteria in NBM and exposed for 24 hours. The resultsindicated that there was no statistical difference between the cellularviability of the bacteria exposed to this concentration of copper andthat of the control wells. Future attachment experiments were performedonly after the 72-hour soaking period to ensure this amount of copperwas not present during attachment assays, however this still providesinsight into the mechanism of action for Cu—BTTri, indicating that theselevels of copper would not explain the observed effect. This concept wasalso tested using triazole ligand(1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene) in powder form at the sameamount that would be present in the chitosan/Cu—BTTri films (0.055 mgtriazole mg⁻¹ chitosan/Cu—BTTri film). This amount of ligand was exposedto P. aeruginosa for 24 hours and the resulting bacteria solution wastested for cellular viability using the CellTiter Blue assay. Resultsfrom this were similar to the copper ion experiment, where there was noobserved decrease in bacteria viability after exposure to triazolecompared to the positive control. Finally, a metal ion chelator thatdisplays selectivity for copper ions (Chelex-100 Resin) was added to thebacterial solution in the presence of the chitosan/Cu—BTTri films toremove any labile or weakly associated copper ions that may becontributing to the observed effect. There was no statistical differencein the amount of viable bacteria attached to the films in the absence orpresence of the metal ion chelator, further giving insight into themechanism of inhibition arising from the intact Cu—BTTri within thechitosan matrix. An initial control experiment was also conducted usingthe metal chelator in the presence of bacteria only to ensure theChelex-100 did not compromise the integrity of healthy bacteria cells.

In addition to testing the individual components of the MOF forantibacterial activity, the surrounding bacterial solution can also betested for cellular viability in addition to quantifying the attachedbacteria onto the films. This does not test individual components ofpotential leachates (as was the case for copper ions and triazolepowder), but rather assesses the entire film solution for presence ofany antibacterial agents. For this assay, aliquots of the bacteriasolution surrounding the films (both chitosan and chitosan/Cu—BTTri)were mixed with the CellTiter Blue reagent to determine cellularviability. Likewise to the copper ion and triazole assays, there was nodecrease in bacteria viability observed for either film (chitosan orchitosan/Cu—BTTri) when compared to the positive control. This ensuresthat leachates do not occur at sufficient concentration to compromisethe integrity of healthy bacteria, further demonstrating the localizedeffect of the film on bacteria rather than the release of antibacterialagents. A summary of the control experiments and subsequent results canbe found in Table 2, in which P. aeruginosa was used with chitosan and10% wt/wt chitosan/Cu—BTTri film components for cellular viability byCellTiter Blue assay (Y=yes; N=no). (n≥3).

TABLE 2 >80% Reduction >80% Reduction in Viable in Viable SampleBacteria in 6 hours Bacteria in 24 hours Chitosan/Cu- Y Y BTTri ChitosanN N Copper ions N N Triazole powder N N Bacterial solution N N

In order to confirm that Cu—BTTri remained intact following thebacterial attachment studies, the structural integrity of thechitosan/Cu—BTTri films was assessed by performing pXRD on the samples.FIG. 4 shows the results of this analysis, where (a) is the pXRDdiffraction pattern of chitosan/Cu—BTTri films prior to beginning thebacterial assays and (b) is the pXRD diffraction pattern after thebacterial assays were performed. The key peaks associated with the MOFafter the bacterial assays match those found from films prior tobeginning the bacterial attachment experiments. This provides furtherindication that the Cu—BTTri remains crystalline and intact throughoutthe attachment experiments.

Additional details of the bacterial attachment studies follow. Thebactericidal activity of the bacterial attachment solution wasdetermined by removing an aliquot of the bacteria solution after theexposure period and tested for bacteria cellular viability using theCellTiter Blue assay. (n=6) The bactericidal activity of the averageamount of copper in solution from the chitosan/Cu—BTTri films (as foundby ICP-AES) was determined by exposing that amount of copper (in theform of copper chloride) to the P. aeruginosa bacterial solution for 24hours. The mass of copper chloride was added to the bacteria solution inNBM and stored at 37° C. After 24 hours, 100 μL aliquots of the controlwells (equal volume of NBM without added copper chloride) and the coppersample wells were combined with 300 μL CellTiter Blue solution. Thewells were analyzed in a similar fashion to the CellTiter Blueattachment assay for bacteria cellular viability. (n=3)

The average amount of triazole present in the chitosan/Cu—BTTri filmswas exposed to P. aeruginosa bacteria solution in a similar fashion tothe copper chloride solution to test for antibacterial activity.Briefly, the average mass of triazole powder was introduced to thebacteria solution in NBM for a 24-hour exposure period. Aliquots of theremaining bacterial solution were combined with CellTiter Blue solutionand the cellular viability was assessed by comparing the wellscontaining triazole to the wells containing NBM only. The triazolepowder in the absence of bacteria was also tested using the CellTiterBlue solution as a negative control to ensure the triazole did notadversely affect the CellTiter Blue reagent. (n=3)

Control studies were also performed using chitosan films with copperchelated to the chitosan. These films were soaked for either 24 or 72hours in NBM at 37° C. and the resulting soaking solution was exposed toP. aeruginosa bacteria solution. The CellTiter Blue assay was performedon the bacteria solution after 24-hour exposure time and thecopper-chitosan soaking solutions were compared to a positive control ofbacteria solution only. (n=4)

The metal chelator Chelex-100 Resin (1-2 mg) was added to wellscontaining the 10% chitosan/Cu—BTTri films before performing the 6- and24-hour attachment studies. An equivalent amount of Chelex-100 Resin wasalso added the bacterial solution in the absence of the films as anadditional control study. (n=3)

Impact of Varying Cu—BTTri Concentration

In order to determine the threshold for the observed reduction inbacterial attachment, a range of MOF compositions were tested, yielding1%, 5%, 10%, and 20% wt/wt Cu—BTTri incorporation. FIG. 5 displays theresults of this assay for all film compositions, with the polystyrenewell again used as the positive control. Average and 95% confidenceinterval are displays. Statistically significant differences betweencellular viabilities are indicted (*) and not statistically significantdifferences are indicated by (ns) as determined by a one-way ANOVA.

After 6 hours of exposure to bacteria, the reduction in attachmentobserved between the chitosan films and the 1% chitosan/Cu—BTTri filmsare not statistically different. Likewise, the films containingadditional incorporation of the MOF (5% and 20% wt/wt) display nostatistical difference observed for all values of cellular viabilitywhen compared to the 10% films. After 24 hours exposure to bacteria, thechitosan and the 1% Cu—BTTri films show no statistical difference inreduction, while the 5% and 20% films are comparable to what wasobserved for the original 10% films. Based on these findings using avariety of MOF compositions, it can be seen that the threshold forbiofilm inhibition begins with the 5% wt/wt incorporation, and thedesired function does not increase as more Cu—BTTri is incorporated intothe chitosan matrix (as seen with both 10% and 20% wt/wt).

Cumulative amount of copper in solution (as percent copper in solutionrelative to total copper content of films) in soaking solutions of 1%,5%, 10%, and 20% wt/wt chitosan/Cu—BTTri films after 72 hours arepresented in Table 3. Soaking studies were performed in NBM at 37° C.over three 24-hour periods. Data presented as average ±standarddeviation, n=3.

TABLE 3 Copper in Copper in Copper in Cu-BTTri solution after solutionafter solution after incorporation 24 h (% original 48 h (% original 72h (% original (% wt/wt) Cu content) Cu content) Cu content) 1 0.10 ±0.03 0.36 ± 0.25 0.76 ± 0.45 5 0.48 ± 0.27 0.96 ± 0.35 1.69 ± 0.69 101.85 ± 0.52 2.25 ± 0.57 2.47 ± 0.59 20 0.22 ± 0.07 0.52 ± 0.12 0.83 ±0.33

The extensive control studies performed using the 10% wt/wtchitosan/Cu—BTTri films gives insight into the factors that do notinfluence the observed antibacterial nature of the films. By ensuringthat the bacterial solution above the films is not adversely affectedcompared to the positive control, it can be concluded that leachatesfrom the films are not acting in an antibacterial fashion. Additionally,it would appear that any labile copper ions not directly coordinatedwithin the material framework are not responsible for the inhibition ofbacterial attachment, as the chelator control experiment would haveremoved those copper ions from solution. Finally, demonstrating theobserved effect for the incorporated Cu—BTTri at different weightpercent's would indicate that there is an upper and lower limit to theamount of incorporated MOF necessary to elicit the desired response.When adding only 1% wt/wt Cu—BTTri, there was no statistical differenceobserved between the chitosan and chitosan/Cu—BTTri with regard to theirinhibition properties. Conversely, increasing the incorporated Cu—BTTrifrom 5% to 10% and 20% wt/wt did not increase the observed antibacterialnature, suggesting a saturation point to the activity imparted onto thebacteria.

A significant reduction (˜85%) in bacterial attachment was demonstratedusing a water-stable MOF blended with chitosan. This inhibition isconsidered substantial in the field of novel antibacterial surfaces,particularly noteworthy for the bacteria strain P. aeruginosa which isknown to be a robust biofilm former. Characterization by ATR-IR and pXRDconfirm the structural integrity of the MOF before and after blendinginto chitosan films. Chitosan/Cu—BTTri blended films were utilized forbacterial attachment inhibition properties by testing the activityagainst P. aeruginosa over 6- and 24-hour periods. The biofilminhibition of P. aeruginosa was observed for the 5, 10, and 20% wt/wtblended films after 6 hours and was maintained for the entire 24-hourchallenge period. This functionality was retained after a second roundof bacterial attachment studies, suggesting reusability of thesematerials as antibacterial surfaces. Finally, extensive control assayswere performed to differentiate this observed antibacterial effect tothe previous antibacterial publications for copper-based MOFs where theproposed mechanism is the slow, continuous release of copper ions. Thesecontrol studies allow us to isolate the observed biological effects tothe chitosan/Cu—BTTri film itself and not to possible leachates fromfilms during experiments. This material presents an opportunity for anovel biomaterial to be utilized as a passive antibacterial surface insettings with prevalent bacterial infections to serve as a biofilminhibitor.

Various modifications and additions can be made to the exemplaryembodiments discussed without departing from the scope of the presentinvention. For example, while the embodiments described above refer toparticular features, the scope of this invention also includesembodiments having different combinations of features and embodimentsthat do not include all of the above described features.

The following is claimed:
 1. A substrate having an antibacterialsurface, the substrate comprising: a chitosan matrix; and water-stablemetal-organic frameworks dispersed throughout the chitosan matrix, thewater-stable metal-organic frameworks present in an amount of 5% wt/wtto 20% wt/wt based on total solids of the substrate.
 2. The substrate ofclaim 1, wherein the water-stable metal-organic frameworks arecopper-based, water-stable metal organic frameworks.
 3. The substrate ofclaim 1, wherein the water-stable metal-organic frameworks areH₃[(Cu₄Cl)₃—(BTTri)₈](H₃BTTri=1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene).
 4. The substrate ofclaim 1, wherein the water-stable metal-organic frameworks arecrystalline after 72 hours in a nutrient broth media.
 5. The substrateof claim 1, wherein the substrate is a biomedical substrate.
 6. Thesubstrate of claim 1, wherein the water-stable metal-organic frameworkspresent in an amount of 5% wt/wt based on total solids of the substrate.7. A method of making a substrate having an antibacterial surface, themethod comprising: dispersing water-stable metal-organic frameworks in achitosan matrix to form a water-soluble chitosan/water-stablemetal-organic framework material, the water-stable metal-organicframeworks present in the water-soluble chitosan/water-stablemetal-organic framework material in an amount of 5% wt/wt to 20% wt/wtbased on total solids of the material; and converting the water-solublechitosan/water-stable metal-organic framework material to awater-insoluble chitosan/water-stable copper-based metal-organicframework material with a buffer solution.
 8. The method of claim 7,wherein the water-stable metal-organic frameworks are copper-based,water-stable metal organic frameworks.
 9. The method of claim 7, whereinthe water-stable metal-organic frameworks are H₃[(Cu₄Cl)₃—(BTTri)₈](H₃BTTri=1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene).
 10. The method ofclaim 7, wherein the water-stable metal-organic frameworks arecrystalline after 72 hours in a nutrient broth media.
 11. The method ofclaim 7, and further comprising forming the water-insolublechitosan/water-stable copper-based metal-organic framework material intoa biomedical device.
 12. The method of claim 7, wherein the water-stablemetal-organic frameworks present in an amount of 5% wt/wt based on totalsolids of the substrate.
 13. A method of using a material to reduceadhesion of bacteria on a surface of the material, the methodcomprising: exposing the material comprising copper-based metal-organicframeworks dispersed throughout a chitosan matrix to a solutioncontaining the bacteria, wherein during the exposure the materialreduces bacterial adhesion by at least 85% in the first six hours ofexposure as compared to material that does not include the copper-basedmetal-organic frameworks and wherein after the first six hours ofexposure the material does not release copper in a bactericidaleffective amount.
 14. The method of using the material of claim 13, andfurther comprising: removing the material from exposure to the bacteria;sterilizing the material after the removing step; and exposing thematerial to a new environment of bacterial after the sterilizing step,wherein during the second exposing step the material reduces bacterialadhesion by at least 85% in the first six hours of exposure as comparedto material that does not include the copper-based metal-organicframeworks and wherein the material is not subject to regenerationbefore the second exposing step.
 15. The method of claim 13 wherein thebacteria is Pseudomonas aeruginosa.